Enhancement of the cellular immune response

ABSTRACT

DTH-Effector cells are primed with carbohydrate antigens and used to enhance the cellular immune response. Tumors have been inhibited by DTH-Effector cells primed with epiglycanin and with synthetic T and Tn antigens. Either the DTH-Effector cells, or these tumor-associated carbohydrate antigens directly, may be used for tumor prophylaxis and therapy.

This application is a continuation of Ser. No. 07/883,234, filed May 7,1992, now abandoned, which is a continuation of Ser. No.07/524,310 filedon May 17, 1990, now abandoned, which is a division of Ser. No.7/222,390, filed on Jul. 21, 1988, now Pat. No. 4,971,795, which is acontinuation of Ser. No. 06/883,266, filed on Jul. 8, 1986 which is nowabandoned.

BACKGROUND OF THE INVENTION

Vertebrates have two basic immune responses: humoral or cellular.Humoral immunity is provided by the special class of cells produced by Blymphocytes. These cells produce antibodies which circulate in the bloodand lymphatic fluid. On the other hand, cell mediated immunity isprovided by the T cells of the lymphatic system.

The cellular immune response is particularly effective against fungi,parasites, intracellular viral infections, cancer cells and foreignmatter, whereas the humoral response primarily defends against theextracellular phases of bacterial and viral infections.

Containment of antigen at its point of entry is accomplished by wallingoff the area by local inflammation. Acute inflammation is characterizedby the influx of plasma proteins and polymorphonuclear leukocytes.Chronic inflammation is characterized by the infiltration ofT-lymphocytes and macrophages. When acute (antibody induced) and.chronic (T-cell induced) inflammations occur in the skin, they arecalled immediate and delayed type hypersensitivity reactionsrespectively.

Epiglycanin (epi) is the major cell surface glycoprotein produced by themammary adenocarcinoma transplantable cell line TA3Ha. Friberg, Jr.,J.N.C.I., 48:1463 (1972); Codington, et al., Cancer Res., 43:4373(1983). The TA3Ha carcinoma cells are covered by a mucin-like glycocalixcomposed mainly of epiglycanin. Codington, et al., J.N.C.I., 60:811(1978); Miller, et al., J.N.C.I., 68:981 (1982). Epi is mainlycarbohydrate in composition, and expresses multiple T and Tndeterminants. T and Tn are general carcinoma autoantigens. Springer,Science, 224:1198 (1984).

Synthetic T and Tn antigens have been prepared, and used in DTHdiagnostics. Lemieuax, EP Appl. 44,188. However, their therapeutic orprophylactic use has not been disclosed.

Bretscher, Eur. J. Immunol., 9:311-316 (1979) cultured DTH-Effectorcells in vitro and injected them with the sensitizing antigen (burroerythrocytes) into the foot pads of mice. We have usedcancer-associated, well characterized carbohydrate antigens to sensitizeeffector cells and have taught a therapeutic use for them.

It is known that tumors may be treated with a mixture of IL-2 andIL-2-activated killer cells. See Fortune, pages 16-21 (Nov. 25, 1985),reporting on the work of Steven Rosenberg. The IL-2 induces a generalimmune response. It thus places a heavy burden on the immune system,diverting resources from the desired cellular response. High, possiblytoxic levels of IL-2 are required to induce a cellular response. Atthese levels, it may unintentionally induce an autoimmune response. Ourprocedure induces a response which is specific, because the cellsrecognize a cancer-associated carbohydrate markers. Only a low level ofour inducer is required to stimulate the cellular immune system.

SUMMARY OF THE INVENTION

We have found that the cell-mediated immune response to cancer cells isenhanced by parenteral administration of natural and syntheticcancer-associated carbohydrate antigens or by parenteral administrationof DTH effector cells primed by such antigens. This enhancement islikely to be advantageous for both therapy and prophylaxis when thebody's primary defense against a challenge is a cellular immuneresponse, e.g., against tumors. The use of other specific carbohydrateantigens, including polysaccharides, glycolipids and glycoproteins, maybe useful in protecting a subject against other threats controlled by Tcells. It is known that parasites, viruses, bacteria and fungi bearsurface carbohydrate determinants.

While we do not favor the use of lymphokines (e.g., IL-2) alone toinduce a general cellular immune response, lymphokines may be used inconjunction with the specific antigens or primed DTH-Effector cells ofthis invention to enhance the specific CMI response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that epi-primed splenocytes induce local DTH swellings inthe presence of epi, while unprimed spleen cells, irradiated tumorcells, and primed cells in the absence of antigen do not.

FIG. 2 shows that this reaction is specific to cancer cell lines bearingthe primer as a surface antigen.

FIG. 3 shows that the reaction was with the T alpha, T beta and Tndeterminants of epi.

FIG. 4 shows that the phenotype of the anti-carbohydrate DTH effectorcells is Thy-1+, Lyt-1+, Lyt-2-.

FIG. 5 shows systemic transfer of DTH by a DTH reaction to irradiatedtumor cells after IV administration of effectors.

FIG. 6 shows inhibition of tumor growth after local transfer ofDTH-Effector cells mixed with tumor cells.

FIG. 7 shows the importance of the priming dose.

FIG. 8 shows that at lower doses of antigen the cellular responsepredominated.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1 Use of S-TAGS to EnhanceCellular Immune Response to Tumors

Synthetic tumor-associated glycoproteins (S-TAGS) and other carbohydrateantigens are known in the art and may be prepared by any convenienttechnique. T and Tn antigens are preferred. For synthetic methods, seeKaifu and Osawa, Carbohydr. Res., 58:235 (1977); Ratcliffe, et al.,93:35 (1981); Paulsen, et al., 104:195 (1982); Bencomo and Sinay, 116:69(1983).

Soluble S-Tags or their aggregates are mixed with an adjuvant forintradermal, intravenous, intramuscular or intraperitonealadministration.

Groups of five mice at a time were primed with specific doses of T-alpha(TF) (Ta), T-beta (Tb) (asialo-GM1 disaccharide) and Tn S-Tags. After2-3 days, they were challenged with 3×10³ live TA3Ha cells. Markedlyincreased survival was noted in mice receiving about 1 ug of syntheticantigen.

It should be noted that TA3Ha is very deadly. The normalpost-transplantation life expectancy of a mouse is only 15-20 days.

EXAMPLE 2 Use of Cultured DTH Effector Cells as Tumor Inhibitors

We obtained mouse primed DTH effector cells by the following procedure.CAF1/J (Balb×A/J) mice from our animal unit were immunized with 30 ugEpi (or 2 ug of any synthetic antigen), in 50% complete Freund'sadjuvant, intraperitoneally, boosted one week later with a similarinjection. 7-10 days after the last injection the animals weresacrificed, their spleens removed and passed through a nylon mesh tomake a single cell suspension. The cells were washed and cultured in aCOSTAR® well at 1.5×10⁷ cells/8 ml. (RPMI+P.S.+10% FCS)/well. Each wellreceived 10 ug epi (or 1-2 ug of any of the synthetic antigens). Thecells were cultured at 37° C. and 5% CO₂ for 6 days and then harvestedby gently dissociating them from the plastic with a rubber policeman.The primed cells were then tested for DTH reactivity with a number ofantigens, and for their ability to inhibit the growth of the TA3Ha cellline in vivo.

To obtain human primed DTH Effector cells, we would use a modificationof the method of Rosenberg, et al. N. Engl. J. Med., 313:1485-92 (1985).Lymphocytes are removed from blood of a living subject byplasmapheresis. The lymphocytes are cultured with a specific antigen,such as cancer-associated glycoprotein (epiglycanin or a T or Tn S-TAG).We prefer use of these more specific primers to the lymphokine (IL-2)used by Rosenberg, to avoid an autoimmune reaction by the IL-2stimulated DTH-Effector cells upon injection into the subject. The humanlymphocytes are cultured in suitable media for 6 days at a preferredconcentration of about 10⁶ cells/ml. The cells are washed to removeantigen. The cells may then be returned to the patient, intravenously,for therapeutic or prophylactic purposes.

We have demonstrated that co-injection of a mammal withepiglycanin-primed DTH Effector cells and antigen (irradiated tumorcells) induced a DTH reaction (FIG. 1). Spleen cells from mice primedand boosted with 30 ug and 20 ug of epi extracted and purified fromTA3Ha ascites fluid and CFA (intraperitoneally administered one and twoweeks beforehand) were cultured at 1.5×10⁷ cells/well with: (a) 10 ugepi/well and (b) no antigen. In addition, unprimed spleen cells werecultured with (c) 10 ug epi/well and (d) no antigen. Cultures wereharvested on the 6th day and 10⁷ cells were injected subcutaneously intothe foot pad of unimmunized mice together with 10⁶ irradiated TA3Hacells. As a control (e), 10⁶ irradiated TA3Ha cells were injected alone.All the DTH values are presented as net DTH swellings, i.e., swellinginduced by primed cells +Ag! - swelling induced by primed cells alone!.The large differential responses demonstrated that immunization with acancer mucin can induce a DTH reaction to those cancer cells which havethe mucin at the cell surface.

Subsequently, we showed that this reaction was tumor-specific (FIG. 2).A DTH effector cell population was generated as in Example I and testedwith a) TA3Ha; b) Epi-M (epi linked to a SEPHAROSE® microspheres); c)mKSA; and d) no antigen. All the DTH values are presented as net DTHswellings, i.e., swelling of primed cells +Ag.!- swelling of primedcells alone!. The result was that the immunization with the epi mucininduced a specific DTH response to the mucin and to the cells which bearthe mucin (TA3Ha) but not against those that do not bear the mucin(mKSA).

We also explored the antigenic specificity of the DTH reaction ingreater detail (FIG. 3). 10⁷ T cells were primed with epi and wereco-injected with a series of potential triggering antigens (TA3Ha, Tn,T-alpha, T-beta, Epi, Fucose ("Fuc"), HSA, BSA, SEPHAROSE microspheres,and no antigen). All soluble antigens were linked to SEPHAROSEmicrospheres and used at 12 ug/foot pad except epi which was used at 50ug/foot pad. All DTH results are net DTH measurements. The experimentshows that the DTH measured following the injection of epiglycanin is inresponse to carbohydrate determinants on S-TAGs which are also known tobe epiglycanin determinants, but not to those carbohydrate determinantswhich are not represented on epiglycanin (e.g., fucose)

In a similar manner, T cells were primed with other antigens (T-alpha,T-beta, Tn) and the cells were co-injected with various potentialtriggering antigens (Epi, Tn, T-alpha, T-beta, Fucose, BSA) with theresults shown in the table below.

SUMMARY OF SPECIFICITY OF DTH RESPONSE

    ______________________________________                                                  TRIGGERING Ag:                                                      PRIMING Ag: Epi   Tn      Ta   T.sub.b                                                                             Fuc  BSA                                 ______________________________________                                        Epi:        +     +       +    +     -    -                                   T.sub.a :   +     +       +    +     -    -                                   T.sub.B :   +     +       +    +     -    -                                   Tn:         +     +       +    +     -    -                                   None:       -     -       ND   ND    ND                                       ______________________________________                                    

To confirm that the reactions we were witnessing were indeed DTHreactions we designed an experiment to check for the cell surfacephenotype of DTH effector cells (FIG. 4) An effector cell population wasprepared as before and treated with a) no treatment; b) anti-Thy 1.2antibodies plus complement; c) anti-Lyt 1.2 plus complement; d) anti-Lyt2.2 plus complement; and e) complement alone. The antigen alone was alsoused as a control. All the values presented are net DTH. Treatment ofthe DTH effector cells with anti-Thy or anti-Lyt-1 antibodies withcomplement kills the DTH effector cells but treatment with anti-Lyt-2plus complement did not kill the DTH effector cells; thus, the phenotypeof the specific anti-carbohydrate DTH effector cells is Thy-1+, Lyt-1+,Lyt-2⁻ --the phenotypic pattern shown by other DTH effector cellsreacting against proteins. See Fujiwara, J. Immunol, 135:2187(September, 1985).

Thy-1 is an antigen found on all mouse T cells. See Roitt, et al.,IMMUNOLOGY FIG. 2.5 (1985). Lyt-1 is a mouse helper T cell antigen of67KD believed to be equivalent to the human antigen detected by theOrtho monoclonal antibody T1. Similarly, Lyt 2 (Ly2) is equivalent tothe human T cell antigen recognized by Ortho monoclonal antibody T8 orBecton Dickinson monoclonal antibody Leu2a.

The designation CD5 is the preferred scientific term for cell typesmarked by the antibodies T1/Leul, or Lyt-1. The designation CD8 is thepreferred scientific term for the cell types marked by T8/Leu2 orLyt-2,3. Thus, the DTH effector cells of the present invention arepreferably described as T cells of the CD5 phenotype and not of the CD8phenotype. See "Note on Nomenclature, Nature, 325:660 (Feb. 19, 1987),adopting the CD nomenclature"; Shaw, "Characterization of HumanLeukocyte Differentiation Antigens," Immunology Today, 8:1 (1987); andLeucocyte Typing (Bernard, ed., 1984) and Leucocyte Typing II (Reinherz,ed., 1986).

The protocol for this comparison was as follows. A primed cellpopulation was obtained, washed, counted, and divided into separatetubes for treatment. The cells were gently pelleted and resuspended inLeibowitz media containing 1) anti Thy 1.2 antibody (at 1/1000 diln.,obtained from NEN), 2) anti Lyt 1.2 antibody (at 1/1000 diln., obtainedfrom NEN) or 3) anti Lyt 2.2 antibody (at 1/1000 diln., obtained fromNEN) at 2×10⁷ cells/ml., and incubated at 4° C. for 45 min. The cellswere then spun down and resuspended in guinea pig complement (1/10diln.) in Leibowitz medium at 2×10⁷ cells/ml., and incubated for 30 min.in a 37° C. water bath. After incubation, cells were washed withLeibowitz + 10% FCS and counted.

A comparative study was undertaken to determine whether the reaction wasof the delayed type. DTH effector cells were injected together withTA3Ha and the reaction was compared with reactions in other miceinjected with only the DTH effector Cells or only the TA3Ha. It wasfound that the reaction peaked in 48 h and had disappeared by 96 h. NoDTH (emphasis on delayed) was found with the other two immunogens.

As further confirmation that the reaction observed was a DTH, amononuclear infiltrate was observed at the site of swelling.

The foregoing experiments all related to local transfer of DTH. We alsoinvestigated the possibility that DTH effector cells would have asystemic effect.

DTH effector cells were administered intravenously to the host animal.Four hours later, irradiated TA3Ha cells were injected into the footpad.At 6, 24, 48 and 72 hours, the DTH reaction was measured (open boxes)and compared with a negative control (plusses) (FIG. 5).

The irradiated tumor cells used in the above-described experiments willnot proliferate. In the next experiments, however, untreated tumor cellswere employed.

DTH primed cells were induced as described above. 5×10⁷ primed cellsplus 5×10⁵ live tumor cells were injected s.c. into the foot pad. Thecontrol mice were injected with normal spleen cells plus tumor cells.The foot pad swelling was typically measured at 12, 24, 48, and 72 hoursand every two days thereafter. To determine the component of theswelling which was due to a DTH reaction, as opposed to a combination ofDTH and tumor growth, mice were injected with primed or normal cellsplus irradiated tumor cells. The swelling was attributed only to the DTHreaction. Tumor growth was measured every 2 days starting 48 hours afterthe peak DTH swelling. The DTH and the tumor size were measured usingcalipers in order to estimate the increase in foot pad thickness.

FIG. 6 shows that the primed cells inhibited the growth of the TA3Hatumor.

Other experiments have shown that the nature of the reaction to theantigen is dependent on the dose of primer administered to theDTH-Effector cells. At lower doses, cell-mediated immunity waspredominant, while at higher doses, the reverse was true.

A dose dependence was also found when splenocytes are primed with Tn inculture (FIG. 7).

We have further shown that the DTH effector cells stimulated by epi will"home in" on a tumor and inhibit tumor growth. DTH-Effector cells wereprimed with epi as previously described. One day prior to intravenousinjection of the epi-primed DTH effector-cells, the TA3Ha tumor wastransplanted into the footpads of mice. Mice with tumors transplanted atthe same time were used as controls and tumor growth was determined bymeasuring the swelling of the footpads (in mm) with calipers. The sizeof the footpad of each mouse at the times stated is given in the tablesbelow:

    __________________________________________________________________________    EXPERIMENT 1                                                                                 Day 6, post iv                                                                        Day 8, post iv                                         __________________________________________________________________________    Systemic Transfer                                                                            10, 12, 12, 10, 15                                                                    50, 15, 80, 80, 50                                     Effector cells                                                                Control Animals                                                                              140, 25, 35                                                                           190, 200, 220                                          (3 mice)                                                                      __________________________________________________________________________    EXPERIMENT 2                                                                  Day 5       Day 6  Day 8   Day 10                                             post iv     postiv post iv post iv                                            __________________________________________________________________________    Systemic                                                                           10, 20, 15, 10                                                                       55, 20, 15, 20                                                                       100, 140, 80, 60                                                                      300, 360, 340, 380                                 Transfer                                                                      Effector                                                                      cells                                                                         (4 mice)                                                                      Control                                                                            60, 75, 60, 110                                                                      170, 160, 170                                                                        340, 300, 320,                                                                        greater than                                       Animals     110    340     500 (3 mice), 410                                  (4 mice)                                                                      __________________________________________________________________________

As may be seen from the above data, the IV-administered, epi-primedDTH-Effector cells exerted a tumor-inhibitory effect at the footpad siteof tumor transplantation.

EXAMPLE 3 Use of Epiglycanin as a Tumor Inhibitor

Epiglycanin (epi) was extracted from the ascites fluid of TA3Hatumor-bearing mice.

Ascites from outbred TA3Ha ip tumors was collected and cells wereremoved by centrifugation. The samples were stored frozen. Beforeextraction, the ascites was thawed and incubated at 37° C. for 2 hours,and any aggregates were removed by centrifugation. The freezing, thawingand clotting procedure was performed twice before peanut a gglutinin(PNA)

25-40 ml. bed volume of peanut a gglutinin (PNA) agarose (E. Y.Laboratories ) was washed with PBS, mixed with ascites and tumbledgently at 4° C. for 24-48 hours. The slurry was poured into a column andextensively washed with PBS at 40° C. with continuous stirring for 24hours. Epi was eluted from PNA agarose using galactose. The elutedepi-galactose mixture was then dialyzed against PBS to remove thegalactose. The sample was lyophilized and stored at -20° C. indessicator.

Groups of five mice were primed with specific doses of epi. After 2-3days they were challenged with 3'10³ live TA3Ha cells. Table 2 belowshows the relationship between the priming dose and survival.

                  TABLE 2                                                         ______________________________________                                                                              No. alive                               Priming dose: Survival: (days)                                                                          ave.   s.e. (at d. 49)                              ______________________________________                                        0.2   ug      19, 25, 27, 49, 49                                                                        33.8   6.3  2/5                                     1     ug      25, 49, 49, 49, 49                                                                        44.2   4.8  4/5                                     2     ug      14, 19, 19, 25, 49                                                                        25.2   6.2  4/5                                     10    ug      20, 22, 22, 25                                                                            22.3   .9   0/4                                     20    ug      20, 22, 25, 49,                                                                           29.0   6.0  1/5                                     100   ug      19, 19, 20, 25, 49                                                                        25.5   5.3  1/5                                     PBS           19, 19, 20, 20                                                                            20     0.3  0/4                                     ______________________________________                                    

Data calculated on day 49 i.e. these mice were still alive. These micewere sacrificed on day 88 to continue the experiment.

Thus, the preferred priming dose was 1 ug, and this dosage appeared toconfer protective immunity against TA3Ha cells.

In another experiment, it was determined- that the humoral response wasnegligible at that dosage (FIG. 8), suggesting that the tumor wasinhibited by a T cell-mediated mechanism.

A summary of our experiences with Epi and T-alpha in tumor inhibition isgiven in Table 3 below:

                  TABLE 3                                                         ______________________________________                                        In Vivo Protection;                                                           Number of animals alive at 4 weeks.                                           Dose of Ag Epi       T-alpha/HSA                                                                             Glu-BSA/HSA                                    ______________________________________                                        0.1     ug     1/20      4/15    0/10                                         0.5     ug     5/20      7/15    0/10                                         1.0     ug     8/20      5/15    0/10                                         5.0     ug     8/15      6/15    0/10                                         10.0    ug     7/20      3/15    0/10                                         cont.          0/20      0/15    0/10                                         ______________________________________                                    

Since G/c-GSA/HSA represents a carbohydrate which is not expressed onthe surface of the TA3Ha tumor cells, administration of that substancedid not provide protection. Epi, which is the predominant surfacecarbohydrate antigen on TA3Ha cells, and T-alpha, which is one of theEpi determinants, did provide protection.

What is claimed is:
 1. A method of enhancing cellular immunity in amammal which consists essentially of administering an immunologicallyeffective amount of a carbohydrate antigenic determinant-bearingantigen, said antigen being a synthetic conjugate of a syntheticcarbohydrate antigenic determinant and a non-tumor associatedimmunogenic carrier not naturally associated with said determinant, saidmammal subsequently exhibiting a cellular immune response elicited bysaid antigen and specific to the carbohydrate antigenic determinant ofsaid antigen, said response being mediated by the DTH-effector cells ofsaid mammal, said cells having a CD5 positive, CD8 negative phenotype.2. The method of claim 1 in which the immunity is immunity to tumors andthe carbohydrate antigen is tumor-associated.
 3. The method of claim 1in which the determinant is the Tn determinant.
 4. The method of claim 1in which the determinant is the T determinant.
 5. The method of claim 2in which the amount of carbohydrate antigen is selected so that thecellular immune response predominates over the humoral immune response.6. The method of claim 2 in which the carbohydrate antigen is aglycoprotein.
 7. The method of claim 1 in which the mammal is a humanbeing.
 8. The method of claim 1 in which the carbohydrate determinant isassociated with adenocarcinomas.
 9. The method of claim 1 in which thecarrier is a protein.
 10. The method of claim 1 in which the conjugateis a glycoprotein.